The complement system is capable of tissue and cell destruction and is therefore a major element of the defense system against invasion by foreign tissue. However, control of this system is necessary in order to prevent destruction of autologous cells. A large number of proteins which are involved in control of the complement cascade have been described.
Most relevant to the present invention is the group which controls the C3 and C5 convertases of both the alternative and classical complement pathways. The target group thus includes serum proteins such as C4-binding protein and factor H and membrane proteins such as C3b receptor, C3d/Epstein-Barr virus receptor, decay-accelerating factor (DAF), and the protein of the invention, membrane factor protein (MRT). MTP inactivates both isolated C3b and C4b and the forms of these proteins as included in the convertases. Reviews of these various factors and their role in the complement cascade regulation can be found in Holers, V. M., et al., Immunol Today (1985) :6:188; Ross, G. D., et al., Adv Immunol (1985) 37:217; Atkinson, J. P., et al., Immunol Today (1987) 8:212; Hourcade, D., et al., Adv Immunol (1989) 45:381-416; Reid, K. B. M., et al., Immunol Today (1986) 7:230.
Much is known concerning these regulatory proteins, which are encoded at a single chromosomal location, the regulators of complement activation (RCA) cluster, except for MCP. They are each composed of multiple repeat of an approximately 60-amino acid consensus sequence composed of conserved cys, pro, gly, trp, leu/ile/val, and tyr/phe residues (Reid, K., et al., Immunol Today (1986) (supra). The genes encoding these proteins have been localized to the long arm of human chromosome 1, band lq32 and form the multigene family designated the RCA gene cluster. As will be shown below, MCP is also a member of this family.
The RCA encoded proteins regulate the complement pathways in two major ways--acceleration of the decay of the C3 convertases crucial to the pathway by reversible dissociation of their component proteins (decay accelerating function) and behavior as a cofactor in the irreversible factor I (a serine protease) mediated proteolytic deactivation of the convertase (cofactor activity), Hourcade, D., et al., Adv Immunol (supra).
A well-studied member of this family related to the MCP of the invention is the decay-accelerating factor (DAF), as recently reviewed by Lublin, D. M., et al., Ann Rev Immunol (1989) 7:35-38. DAF is present on virtually all peripheral blood cells, including erythrocytes, granulocytes, T and B lymphocytes, monocytes, and platelets; in addition, soluble forms of DAF have been found in extracellular fluids and tissue culture supernatants. The gene encoding DAF has been cloned and sequenced by two groups: by Medof, M. E., et al., Proc Natl Acad Sci USA (1987) 84:2007-2011; and by Caras, I. W., et al., Nature (1987) 325:545-549. Two classes of DAF cDNAs have been found (Caras et al., Nature (supra)). The difference between the two forms is the addition of 118 bp near the carboxy terminus of one form; this insert resembles an Alu type of sequence and its internal boundaries match the intron consensus splice sequences. This has lead one group (Caras et al.) to postulate that this class of cDNAs include an unspliced, retained intron. The suggestion by Caras that the membrane and soluble secreted forms of DAF result from differential splicing of the mRNA to include an intron is also described in PCT application WO89/01041. It has been found by the inventors herein that the inserted sequence is encoded by exon 10 of the DAF gene, and that this exon is flanked by consensus splice junction sequences (Post et al., J Immunol (1990) 144:740). Therefore, the etiology of the two classes of DAF-encoding cDNAs is conventional alternative splicing of a distinct exon.
MCP was initially identified by iC3/C3b affinity chromatography on surface-labeled peripheral blood cells and designated gp45-70 to describe the range of M.sub.r obtained on SDS-PAGE (Cole, J. L., et al., Proc Natl Acad Sci USA (1985) 82:859). MCP was partially purified from the human mononuclear cell lines and shown to have a cofactor activity but no decay accelerating function (Seya, T. J., et al., J Exp Med (1986) 163:837). MCP is absent from erythrocytes, but present as a membrane-bound protein on human T and B lymphocytes, granulocytes, monocytes, platelets, endothelial cells, epithelial cells, and fibroblasts (Seya et al., Eur J Immunol (1988) 18:1289; McNearney, T., et al., J Clin Invest (1989) 84:538). The occurrence of MCP on a wide range of host cells is consistent with a role in protecting host cells from damage by complement (Hourcade, D., et al., Adv Immunol (supra); Lublin, D. L., and Atkinson, J. P., Current Topic in Microbial and Immunol (1989) 153:123-145). On most of these cells it occurs in two forms of molecular weight 63 kd and 68 kd, as determined by SDS-PAGE. The quantity of each of the two species expressed is under genetic control and involves a two allelic system (Ballard, L., et al., J Immunol (1987) 138:3850-3855). The MCP found by immunoprecipitation on the membranes of granulocytes appears, however, not to exhibit this polymorphism (Seya, T., et al., Eur J Immunol (1988) 18:1289-1294).
In addition to human MCP, MCP or MCP-like or MCP-related materials have been found in a variety of mammalian tissues. For example, a dimorphic protein of MW 65 kd and 69 kd is found on orangutan erythrocytes which binds to homologous C3, is immunoreactive with a monoclonal antibody raised against human MPC, and has cofactor activity, as described by Nickelis, M. W., et al., (1990) submitted. Both marmoset and rabbit also exhibit dimorphic proteins of 75 and 68 kd and of 55 and 45 kd from erythrocytes and platelets, respectively, which bind C3 (Goujet-Zalc, C., et al., Cellular Immunol (1987) 109:282; Manthei, U., et al., J Immunol (1988) 140:1228). In addition, erythrocytes of baboon, most cells in mice, and alveolar and peritoneal macrophages of rabbit produce a 65 kd protein which is capable to bind to C3 (Birmingham, D. J., et al., J Immunol (1989) 142:3140; Wong, W. W., et al., J Immunol (1985) 134:4048; Schneider, R. J., et al., Nature (1981) 190:789; Cui, W., et al., FASEB Journal (1989) 3:A500).
The previously purified human MCP has been utilized to prepare a polyclonal rabbit antiserum monospecific for this protein. The antisera were raised in rabbits by repetitive injections of MCP purified as described by Seya, T., et al., J Exp Med (1986) (supra), in complete Freund's adjuvant. These antisera have been used to identify MCP in extracts from various membranes.
The present invention provides a more highly purified form of this protein and the capacity to produce it recombinantly, thus providing practical quantities for therapeutic use. In addition, as shown hereinbelow, the MCP protein may be made recombinantly in a variety of forms with varying capacity for glycosylation and membrane binding, thus permitting regulation and optimization of therapeutic forms.